Compositions and methods for detecting single gene copies in-situ

ABSTRACT

Compositions and methods are provided for detecting single copies of a gene in-situ using brightfield microscopy in detection of various target nucleic acid sequences, such as genes or nucleic acid of microorganisms in a cell. In particular, nucleic acid probes for various target nucleic acid sequences in a cell are utilized in conjunction with enzymes and their substrates to produce chromogenic composition associated with the target nucleic acid sequences in the cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 09/863,125,filed May 22, 2001, which is a continuation of Ser. No. 09/419,421,filed Oct. 15, 1999, abandoned, which are incorporated herein byreference in their entirety, and to which applications we claim priorityunder 35 USC §§ 120 and 121.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to techniques for detection andlocalization of nucleic acid and, more particularly, to the use ofenzyme catalyzed chromogenic compositions in such detection andlocalization.

2. Description of Related Art

In-situ hybridization (ISH) techniques are an important tool fordetection of nucleic acid sequences, i.e., both DNA and RNA. Uniquenucleic acid sequences occupy precise positions in chromosomes, cellsand tissues and in-situ hybridization allows the presence, absenceand/or amplification status of such sequences to be determined withoutmajor disruption of the sequences.

It is known that certain nucleic acid sequences are associatedpathologic conditions in living organisms. For example, the presence ofcertain genes and viral nucleic acids have been implicated inprecancerous and cancerous pathology. Genetic diseases are alsodiagnosed by determining the presence, absence or number of copies ofnucleic acids. Several genetic markers have been associated with poorprognosis in patients with various cancers. Infectious microorganisms,particularly intracellular ones, contain nucleic acid sequences whichare also detected to diagnose disease and monitor therapy.

Accumulation of alterations in both cellular oncogenes and tumorsuppressor genes has been associated with human tumorigenesis. Geneamplification has been associated with certain aggressive forms of humancancer and has been used as a prognostic parameter in the clinicalanalysis of certain malignancies. Presence or absence of nucleic acidamplification can also be used to indicate treatment in certain cancersof disease states. Cellular oncogene amplification of the HER-2/neuoncogene has been shown to play an important part in the pathogenesisand prognosis of various solid tumors including breast cancer. See,e.g., Battifora et al., Modern Pathol, 4:466-474 (1991) and Press etal., Cancer Res., 53:4690-4970 (1993).

Loss and/or mutation of tumor suppressor genes is also indicative ofcertain cancers and certain stages of cancers. The loss of p53 isclassically found in many solid tumors. In such a situation a cellusually has only one mutated copy of the gene, the other copy being lostdue to aneuploidy. Loss of both copies is also found.

Human Papilloma virus (HPV) is a common sexually transmitted viraldisease. There are at least 70 distinct types of HPV. Some HPV typesfound in genital lesions have been implicated in cervical precancers andcancers (for example, types 16, 18, 31, 33 and 35) while other types arerelatively benign (Types 6, 11, 42, 43 and 44).

Currently, Pap smears are performed yearly on women to check for thepresence of atypical or cancerous cells. Roughly 90% of all Pap smearsare normal, 3% are unequivocally dysplastic, and 7% are squamous atypias(ASCUS) or low grade squamous intraepithelial lesions (LSIL). The ASCUSand LSIL diagnoses present the doctor and patient with multiple choicesfor treatment. The ability to accurately test these patients for highrisk type HPV presence would provide further information on the bestcourse of therapy. For example, the presence of a low risk HPV type mayindicate no further action except perhaps more frequent Pap smears. Ahigh risk HPV type presence would indicate a more aggressive approach.

Other viral diseases are also frequently difficult to detect ordistinguish clinically. Examples include Epstein-Barr virus (EBV),cytomegalovirus (CMV), hepatitis viruses, etc. Nucleic acid baseddetection systems performed in-situ for these viruses is also desirable.

At least four types of nucleic acid probes are commonly used for in-situhybridization. These include double stranded DNA (dsDNA) probes, singlestranded DNA probes (ssDNA), single-stranded RNA probes (ssRNA), andoligonucleotide probes. The production and application of a largevariety of DNA and RNA probes has been made possible through theavailability of many molecular cloning techniques including plasmid,phage P1, cosmid, and yeast artificial chromosome (YAC) cloningprocedures, cell hybrid technology, chromosome sorting and dissectiontechniques, and amplification techniques such as the polymerase chainreaction (PCR). Additionally, the use of DNA synthesizers can permitoligonucleotides to be custom designed and chemically synthesized.Different target sequences such as specific genomes, chromosomes,repetitive and unique sequences, microsatellites, mitochondrial nucleicacids, mRNA, or microbial (viral) nucleic acids may be identifieddepending on the selection of probe used in the ISH procedure. Nucleicacid probes may be labeled by conjugation to a marker to create adetectible probe hybridization site.

A variety of detection systems have been developed which are based onligands which bind to a probe either directly or indirectly and markersor labels which allow visualization of the probe and hence, the sitewhere the probe has hybridized. Radioactive labels or non-radioactivefluorescent labels have been employed as such markers or labels eitherdirectly linked to the probe or attached through secondary means such asantibodies. Although radioactive labels are effective, they areassociated with radioactive toxicity and environmental concerns.Fluorescent non-radioactive detection protocols provide severaladvantages for in-situ hybridization, including easy and rapiddetection, high sensitivity with low endogenous background, highresolution, multiple-target analysis with different fluorochromes, andthe possibility to quantitate signal. Unfortunately, the signalgenerated by fluorescent markers typically fades over time. Uponexposure to light and autofluorescence of the tissue sample may mask thepresence of a target signal. Additionally, the cost and availability offluorescent microscopy equipment and trained personnel is greater thanconventional brightfield microscopy.

Alternatively, enzyme systems have been used for detection of nucleicacid target sequences. Enzymes such as horseradish peroxidase oralkaline phosphatase can be chemically conjugated to proteins,antibodies, avidin, streptavidin, biotin, Fc-binding proteins such asprotein A or G for use in hapten interactions, or directly to thenucleic acid probes. Certain enzymes interact with chromogen substratesolutions to produce distinctly colored products which are capable ofbeing visualized directly through brightfield microscopy. This permitsthe localization of hybridization sites through enzyme precipitationreactions. Some advantages of cytochemical detection with enzymesinclude the stability of the precipitate, indicating permanent storageof cell preparations, and the use of a standard brightfield microscopein a setting where routine analysis is performed.

Oxidoreductases are enzymes which catalyze the oxidation of varioussubstrates, and are well suited for the preparation of enzyme-conjugatesdue to their excellent stability and their ability to yield chromogenicproducts. Peroxidases have been widely used as a label for antibodiesand ligands, such as avidin and streptavidin, in immunoassay systems.Peroxidases catalyze the hydrogen peroxide oxidation of certain electrondonors by transferring electrons from the donor to the peroxide andresulting in formation of a colored product and water.

A number of different chromogens have been used with enzyme-linkedimmunoassays (ELISA). See, e.g., U.S. Pat. No. 4,962,029. A number ofother different enzymes such as kinases and phosphatases catalyze theaddition and removal of phosphate moieties. These enzymes have also beenused in various immunoassays.

TMB has reportedly been used in detection of repetitive DNA sequences inSpeel et al., Rapid Bright-Field Detection of Oligonucleotide PrimedIn-Situ (PRINS)—Labeled DNA in Chromosome Preparations and Frozen TissueSections, Biotechniques, 20:226-234 (February 1996). The PRINS procedureinvolves placing haptens at a target site using enzymes to incorporatelabeled nucleotides by elongating unlabeled primers. More specifically,unlabeled DNA primer is annealed to its complementary target sequencein-situ. The primer serves as an initiation site for chain elongationusing DNA polymers and fluorochrome-, biotin-, or digoxigenin-labelednucleotides. The labeled DNA chain is then detected directly byfluorescence microscopy or indirectly by fluorochrome-conjugated avidinor antibody molecules. Speel et al. describes localization of DNA targetsequences using PRINS and colored precipitates of horseradishperoxidase-diaminobenzidine (brown color), alkaline phosphatase-Fast Red(red color) and horseradish peroxidase-tetramethylbenzidine (greencolor). Results were evaluated using bright-field microscopy.

In Speel et al., A Novel Triple-color Detection Procedure forBright-field Microscopy, Combining In-Situ Hybridization with ImmunoChemistry, J. Hist. Cyt., vol. 47, No. 10, pp. 1299-1307 (1994), aperoxidase-TMB product was detection using in-situ hybridizationtechniques. The system described by either Speel et al. publicationabove was used only for the detection of satellite or repetitive DNA.However, the sensitivity required for detecting such multiple copysequences is much lower than the sensitivity required for detection ofunique copy sequences. If the sensitivity of techniques using labelednucleic acids and their detection systems can be increased to allowdetection of unique copy sequences, more accurate results could beobtained for detection of such sequences in diagnosis andprognostication of cancer or other disease states.

A number of patents have proposed various colormetric determinations ofhybridization. These include U.S. Pat. Nos. 5,851,764, 5,846,728,5,525,465, 5,677,440 and 5,474,916. However, none of these were able todistinguish single gene copies in-situ.

SUMMARY OF THE INVENTION

A method of detecting a target nucleic acid sequence is provided whichincludes hybridizing a nucleic acid sequence to a target; associating anenzyme with the nucleic acid sequence; contacting the associated enzymewith a chromogen substrate composition thereby forming a coloredprecipitate (chromogenic product); and observing the location of thecolored precipitate. The alkaline enzyme may be associated with thenucleic acid sequence by conjugating a hapten to a nucleotide to form ahapten conjugated nucleotide; conjugating enzyme to a binding partner ofthe hapten to form an enzyme conjugated binding partner; and contactingthe enzyme conjugated binding partner with the hapten conjugatednucleotide to form a binding partner/hapten complex.

Visualization is typically done by brightfield microscopy (for examplein in-situ hybridization), spectrophotometry (for example in solutionhybridization) or by visual observation (for example in solid phasehybridization).

A composition is provided which includes nucleic acid associated withthe product of an insoluble chromogen formed by the action of theenzyme, particularly a dephosphorylated product of a alkalinephosphatase and a composition containing an aqueous solution ofNBT/BCIP, a salt, surfactant and buffer.

A method of detecting a nucleic acid sequence is provided which includesobserving the location of the insolubilized enic product associated withthe nucleic acid sequence, particularly in situ.

Using such a system, single copies of target nucleotide sequences may bedetected in a single cell. The method provides for counting 1-5 andgreater numbers of target copies per cell. The location within a cellwhich harbors the target may also be determined.

A primary goal of the present invention is to quantitatively determinethe number of target nucleotide sequences in such a fashion that one maydetermine the number of copies per cell. Clinically, the presence ofmore or less than two copies may be indicative of certain diseases orconditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a microphotograph made according to the present inventionusing brightfield microscopy of detection of an unamplified (normal)number of HER-2/neu gene copies in cells.

FIG. 2 depicts a microphotograph made according to the present inventionusing brightfield microscopy of detection of a low amplified number ofHER-2/neu gene copies in cells.

FIG. 3 depicts a microphotograph made according to the present inventionusing brightfield microscopy of detection of a high amplified number ofHER-2/neu gene copies in cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

By judicious selection of enzyme, chromogen and conditions, one can notonly detect the presence of a nucleic acid target in a sample in-situ,but one can even count individual copies within a cell. While such hasbeen done previously with a fluorescently or radioactively labeledprobe, brightfield detection presents particular problems which hasheretofore prevented counting of individual gene copies. While thesignal emitted from a radioactive or fluorescent compound is inherentlylocalized, an enzyme label is not directly observable. Rather the enzymemust convert a compound in solution into a colored compound which mustnot color the entire region, yet be able to be readily observable.Unlike observation of a radioactive or fluorescent signal from a darkbackground, under brightfield observation, cells naturally containdarkened regions. Thus, in brightfield detection, one is trapped betweenthe limits of delectability and the formation of a spot so large thatcounting of individual gene copies is not possible.

Use of NBT/BCIP, salt, surfactant, and buffer provides a surprisingincrease in sensitivity for detection of nucleic acid sequences usingalkaline phosphatase catalyzed detection techniques. Such increase insensitivity allows detection of unique copy sequences contained within acellular genome that were previously undetectable using TMB basedreagents. It is contemplated that a NBT/BCIP composition according tothe present invention can be used in any technique involvingdephosphorylation and formation of a colored composition to locate anucleic acid sequence. Such techniques include in-situ hybridization(unamplified) and primed in-situ (PRINS) labeling.

The amounts of NBT/BCIP, salt, surfactant and may be varied by routineexperimentation to provide maximum solubility of each ingredient in thecomposition, maximum handling characteristics, e.g., viscosity, andmaximum color dynamics. The amount of NBT in the final reaction maypreferably range from about 200 mg/l to about 2500 mg/l, more preferablybetween about 500 mg/l and about 1300 mg/l and even more preferablyabout 830 mg/l. The amount of BCIP in the final reaction may preferablyrange from about 50 mg/l to about 1500 mg/l, more preferably betweenabout 150 mg/l and about 550 mg/l and even more preferably about 287mg/l. The amount of surfactant may preferably range from about 0.03% toabout 0.001% and is more preferably about 0.015%. The concentration ofmagnesium chloride may range from about 0.15 M to 0.01 M, morepreferably about 0.1 M to 0.06 M, most preferably about 0.08 M. The pHof the composition may be anywhere in the range which permits the enzymeto function without degrading the other reagents or solubilizing thecolored product. The preferable range is from about 7 to about 11, morepreferably about 9.5.

Suitable surfactants include ionic and non-ionic detergents such asdioctyl sulfosuccinate, sarkosyl, cholic acid, Triton X-100 30, Brij-35,deoxycholate, sodium dodecyl sulfate and the like. Polyhydric alcoholsincluding propylene glycol, dipropylene glycol, 1,2,6 hexanetriol, 1,2,4butanetriol, and the like may also be added. A preferred composition iscommercially available and sold by Ventana Medical Systems, Inc.,Tuscon, Ariz.

For other enzyme/chromogen combinations, the amount of TMB maypreferably range from about 0.16 mg/l to about 0.28 mg/l and is morepreferably about 0.22 mg/l. The amount of sodium tungstate is preferablyless than about 25 ppm and more preferably less than about 1 ppm. Theamount of heavy metal salt other than sodium tungstate is less thanabout 25 ppm and more preferably less than about 1 ppm. The amount ofanionic surfactant may preferably range from about 0.06 mg/l to about0.1 mg/l and is more preferably about 0.08 mg/l. The amount ofpolyhydric alcohol may preferably range from about 0.075 mg/l to about0.125 mg/l and is more preferably about 0.1 mg/l. The amount of aproticsolvent may preferably range from about 0.18 mg/l to about 0.31 mg/l andis more preferably about 0.25 mg/l. The pH of the composition shouldpreferably range from about 5 to about 6.5 and is more preferably about5.67.

A preferred heavy metal salt other than sodium tungstate is cobalt salt.Suitable cobalt salts include, acetate, chloride, sulfate, nitrate andthe like. Suitable anionic surfactants include dioctyl sulfosuccinate,sarkosyl, cholic acid, Triton QS 30, Triton X 100, deoxycholate, sodiumdodecyl sulfate and the like. Suitable polyhydric alcohols include 1,2,6hexanetriol, 1,2,4 butanetriol and the like. Suitable aprotic solventsinclude dimethyl sulfoxide, dimethylformamide and the like. A preferredcomposition is commercially available and sold under the trade name TrueBlue Peroxidase Substrate by Kirkegaard & Perry Laboratories (KPL),Gaithersburg, Md.

Differing concentrations, salts, surfactants and other choices ofingredients may also be chosen by routine experimentation provided thatthe result is scored. With different enzyme/chromogen combinations, itis expected that some routine experimentation will be needed tosufficiently optimize the assay to become quantitative.

In general, ISH is well-known and in the present invention involves:

(1) Identification, selection, and labeling of a probe of interest.Suitable probes include oligonucleotides, plasmids, cosmids, yeastartificial chromosomes (YACS), bacterial artificial chromosomes (BACS),P1, viral sequences, etc. Examples of haptens for probe labeling includebiotin, digoxigenin, dinitrophenyl, phenyloxazalone, fluorescent labelsfor direct or indirect labeling include fluorescein, rhodamine,coumarin, Texas Red, Rhodol Green, etc. Incorporation of thehapten-conjugated nucleotides into the nucleic acid sequence of theprobe may be performed by nick translation, PCR, random priming orsimilar means familiar to those skilled in the art.

(2) Preparation of cells and tissue sections for ISH which routinelyinvolves fixation and attachment of the specimen to a solid support suchas a glass slide. Known procedures include formalin fixation, paraffinembedding, freezing, alcohol fixation, cutting, mounting, etc.

(3) Pretreatment of the specimens to permit penetration of the labelednucleic acid probes and cytochemical detection molecules, or to reducenon-specific backgrounds which may be necessary for a given specimen,e.g., proteolytic digestion, RNase treatment, endogenous enzymeinactivation, etc.

(4) Heat, salts, chaotrophic agents such as formamide and/or alkalinetreatment to render the double-stranded DNA probes and targetssingle-stranded as a prelude to,

(5) hybridization, which provides the necessary conditions for the probeto bind to the target nucleic acid. A number of physiochemicalconditions are known that affect hybridization, kinetics andsensitivity. Common conditions affecting rate and sensitivity ofhybridization are the factors associated with stringency, especiallytemperature, salt concentration, chaotrophic concentration, probe lengthand concentration. These parameters are routinely empirically optimizedby those skilled in the art.

(6) Following hybridization, postwashing to remove any nonspecific orlow homology bound probe.

(7) Detecting the presence of labeled probe bound to the in-situ nucleicacid target by incubation of a color forming composition herein with aenzyme linked to, e.g., any of the following: nucleic acid probe,antibody, hapten conjugate, biotin, avidin, steptavidin, or polypeptidein any combination or order.

(8) Following detection, the specimen may be counterstained to providecolor contrast with the signal color and as a means for viewingmorphology of the specimen.

(9) The slide may then be made permanent by covering the specimen with anoninterferring mounting medium and a coverslip.

The ISH process has been automated with equipment from variousmanufacturers. The same equipment with appropriate changes to theprocessing conditions as set forth in this specification may be used forpreparing slides for the present invention using quantitativebrightfield detection.

It will be appreciated that for each target being detected and eachdifferent tissue type, differing concentrations and conditions willlikely be required. The skilled artisan will realize this and performroutine experimentation to optimize the assay for each differing sampleand target.

Various labeling techniques based on chemical or enzymatic modificationof nucleic acid probe sequences may be utilized, e.g., enzymaticincorporation (e.g., nick translation, random priming), incorporationduring DNA amplification (e.g. PCR) or nucleotides conjugated to haptensincluding biotin, digoxigenin, and dinitrophenyl. Incorporation oflabeled nucleotides can also be accomplished with the primed in-situ(PRINS) technique which utilizes an oligonucleotide primer for chainelongation catalyzed by a DNA polymerase or RNA reverse transcriptase.

After hybridization of the probe containing one or more haptenconjugated nucleotides to a target nucleic acid sequence or aftergeneration of a nucleic acid strand incorporating one or more haptenconjugated nucleotide which is complementary to one strand of the targetnucleic acid sequence by PRINS, an enzyme is then associated with thetarget nucleic acid either directly or indirectly. Thus, when either theprobe or the PRINS generated strand duplexes with the target sequence,the incorporated hapten is available for complexation with a bindingpartner. Typically, a binding partner of the hapten is conjugated to theoxidizing enzyme such as peroxidase. Binding partners includepolypeptides, antibodies, avidin, streptavidin and biotin.Alternatively, an oxidizing enzyme can be conjugated directly to theprobe or PRINS generated complementary strand.

After association of the target nucleic acid sequence with the oxidizingenzyme, the specimen is contacted with a composition including, colorforming compound, salts and surfactant in accordance with the presentinvention. The enzyme catalyzes the composition including the colorforming compound to provide an insoluble colored composition, which,after optional counterstaining, can be viewed with a brightfieldmicroscope.

Sample preparation is important to obtaining successful results. Becausethe probe/antibody/enzyme complex or individual components arerelatively large, the cell must be adequately digested to permit thereagents to associate at the location of the chromosome. However, overdigestion is equally problematic as the cell boundaries are unclearmaking the counting of spots/cell difficult. Maintaining cellularmorphology is also important for proper visualization.

In order to count discrete spots of precipitated colored compound, thetype and amount of enzyme label and chromogen is particularly important.Certain chromogens will not be sufficiently localized to form discretespots. For measuring genomic DNA, usually two copies and no more thanfour copies (just before cell division) of any so called “single copy”gene are normally present. The present invention produces spotssufficiently small that at least five copies of any target may bevisualized in a cell. Larger numbers of target may merge together andstill provide adequate identification that this cell is abnormal. Thepresent invention is designed so that there is a one to one correlationbetween spots and copies of the target sequence at a normal copy number.

It is also contemplated that a NBT/BCIP or other chromogen compositionaccording to the present invention can be used for signal amplificationtechniques well-known in the art. For example, multiple binding sitesallow multiple alkaline phosphatase or other enzyme molecules to beassociated with a target nucleic acid thus creating a network ofalkaline phosphatase sites. In this manner, an antibody having multiplebinding sites can be conjugated to a nucleotide associated with a targetnucleic acid sequence. Multiple binding partners conjugated to alkalinephosphatase will then associate with the multiple binding sites of theantibody to provide multiple alkaline phosphatase enzyme molecules.Other techniques for signal amplification are well-known as well. Thus,multiple alkaline phosphatase sites will increase efficiency ofdephosphorylation of NBT/BCIP composition according to the presentinvention.

While the invention's best mode is exemplified by using the combinationof alkaline phosphatase and NBT/BCIP, which gave the best quantitativeresults, the following combinations have also been found to be operableby experimental data in ISH.

Horseradish peroxidase as the enzyme and 3,3′,5,5′ tetramethylbenzidineas the color forming compound in a composition containing, sodiumtungstate and a heavy metal salt other than sodium tungstate, anionicsurfactant, polyhydric alcohol and aprotic solvent has been used todetect single gene copies in-situ. Also, horseradish peroxidase as theenzyme with diaminobenzidine as the color forming compound has been usedto detetct single gene copies in-situ.

It will be appreciated that other enzyme and chromogen combinations maybe used. Suitable lists of possible enzyme and chromogen combinationsare known from the immunoassay field, particularly inimmunohistochemistry. Common examples include horseradish peroxidase andAEC or any of a number of benzidine and napthol based compounds. Any ofthe enzyme chromogen combinations may be amplified using any of thetyramide based systems known per se. Likewise, enzyme and chromogencompositions may be optimized using similar reagents and conditions ashave been used in such immunoassays.

However, unlike immunohistochemistry, single copies of nucleic acids arebeing detected with ISH. In immunohistochemistry, one is almost neverdetecting a single molecule of protein and certainly cannot countindividual molecules in a multi protein molecule per cell sample. Thusone cannot directly utilize any enzyme/chromogen combination fromimmunohistochemistry without first altering the assay in a manner inaccordance with the present invention to generate a quantitative ISH.

While described for in-situ detection, the present invention may be usedto determine the presence of nucleic acid sequences in otherenvironments as well such as extracted DNA and RNA for any hybridizationassays such as Southern, Northern, dot and reverse dot blots.

Levels of mRNA may be detected and even quantified by using the labelingand detection system of the present invention. This detection may beperformed in-situ or by extracted RNA or its cDNA.

The gene HER-2/neu gene is amplified in a number of cancers includingbreast, prostate, ovarian, endometrial and colorectal. In eachsituation, it is important to determine the approximate number of genecopies in each cell. Knowing an average number is unacceptable as a fewcancer cells in a small metastasis in a sample of mostly normal cellsmay have “on average” a “normal” number. Thus, the importance ofcounting the gene copy number within a single cell.

Localization of a target nucleotide sequence within a single cell candetermine whether the target is within the nucleus, mitochondria orassociated with a structure (e.g. the rough endoplasmic reticulum) inthe cytoplasm. This is a different localization from what has beenreferred to in the prior art. The present invention is detecting a spotlocalized in a cell, not a cell or group of cells in a tissue.

In addition to the numerous uses stated above for an in-situ assaydetecting individual gene copies in a cell, the present invention may beused for all genetic testing, including prenatal testing of amnioticfluid, CVS, fetal cells in maternal blood, etc. Genetic testing has usesbeyond disease diagnosis, such as compatibility and tissue typing forblood, tissue and organ transplantation, monitoring bone marrowtransplants, paternity, forensic and archeological testing. Perhaps thebest known example of disease associated with extra copies of a nucleicacid is Down's Syndrome which is detectable by having an extrachromosome 21.

The detection of individual copies of target nucleic acid sequencespermits one to detect aneuploidy, microsatellite instability, lengths ofrepeats (such as in severity of Fragile X determination), susceptibilityto cancer or other diseases and heterozygosity of any gene.

While detecting individual target copies of an infectious microorganismmay not always be necessary, the infection of a virus and itsreplication may be observed and quantified using the present invention.For example, HIV, HSV, CMV, T. palidium, M. tuberculosis, etc.Furthermore, microorganisms may be in a latent or repressed state invery low copy numbers such that a single gene copy per cell may be allthat is present. Microorganisms which integrate into the host cellchromosome may have only one copy per cell present. Thus the greatsensitivity of the present invention is preferred.

One may use multiple probes, each labeled with the same or differentenzymes or haptens for associating an enzyme with the probe. Sucharrangements would permit detection of multiple target sequencessimultaneously. This may further characterize a particular disease stateor detect plural diseases simultaneously. This variation is particularlyuseful when the ratio of one target sequence to another target sequenceis relevant. By using different chromogens, different colored spots maybe formed which can be visualized and counted.

Unlike previous methods of in-situ detection, clinical testing,particularly automated clinical testing involves biological samples froma wide variety of fluids and tissues. A method for detection ofindividual nucleic acid copies in a cell under such diverse conditionsrequires a flexible and superior detection system. The present inventionhas achieved such a result in an automated format.

The following examples are included for purposes of illustrating certainaspects of the invention and should not be constructed as limiting.

EXAMPLE 1 HER-2/Neu Brightfield Assay for Embedded Breast TissueSpecimens

Paraffin embedded tissue specimen slides were baked for 1 to 20 hours at65° C.+/−2° C. The slides were then deparaffinized in three washes ofxylene for 5 minutes each and washed in 100% ethanol, 2 times for 2minutes each and air dried label end down, propped at an angle.

Next, the specimens were digested in 0.5 mg/ml Proteinase K/2×SSC (pH7.0) at 45° C. (Proteinase K solution was prepared by adding 1 mldeionized water to a 25 mg vial of Proteinase K, shaking the vial tosuspend the Proteinase K and transferring the 1 ml of solution to 50 mlof 2×SSC prewarmed to 45° C.). Cell line controls were digested for 12minutes, and tissue specimens for 35 minutes. The slides were rinsed for10 seconds in 2×SSC at room temperature followed by dehydration in aroom temperature ethanol series of 70%, 80%, and 100% ethanol for 1minute each. The slides were air dried with the label end down.

10 μl digoxigenin-labeled probe, prewarmed to 37° C. for 5 minutes, wasadded to each specimen. The slide was then heated at 90° C. for 12minutes (for example, in an MJ Research thermocycler with a slide block)to denature probe and target DNA. The slide was coverslipped andtransfered to a prewarmed humid chamber and hybridized for 4 hours toovernight at 37° C.

The glass coverslip was removed and the slide washed in a Coplin jarcontaining prewarmed 1×SSC for 5 minutes at 72° C. The slides weretransferred to a coplin jar of room temperature wash solution of adetergent and a buffer for example, Tris buffer and Brij-35 (APK washsolution, Ventana Medical Systems, Inc.).

Anti-digoxigenin Alkaline Phosphatase (Boehringer Mannheim Cat. 1093274)was diluted 1:150 in 100 mM Tris pH 7.5/150 mM NaCl. 100 μl of thediluted antibody was added to each slide, covered with a plasticcoverslip, and incubated for 30 minutes at 37° C. in a humid chamber.The slides were then washed in a 10-second stream of APK and transferredto a Coplin jar of APK until all slides are washed.

While the slides drain, a magnesium chloride solution (Ventana Enhancer1:4) (100 ul+300 ul) with APK was prepared. 400 ul was added to eachslide, the slides placed on the Lab-Line Orbit Shaker at roomtemperature and shaken at 1000 rpm for 4 minutes.

Ventana NBT (6×NBT) was mixed 1:1 with Ventana Blue BCIP (6×BCIP) (100ul+100 ul). 200 ul of the mixture was added to the puddle of solution oneach slide and shaking continued for 30-60 minutes. This shaking wasstopped when the reagent pool starts to darken or the development lookscomplete when viewed with the scope. Each slide was washed in a10-second stream of deionized water from a squirt bottle and transferredto a Coplin jar containing deionized water. The deionized water waschanged and incubated for 1 minute at room temperature.

To enhance visualization, the slides were counterstained by draining theslides well and added to a Coplin jar containing 1×KPL Eosin(Cat.71-02-00), diluted from stock in deionized water. Incubation wasfor 20 seconds at room temperature. Each slide was washed in a 10-secondstream of deionized water from a squirt bottle and transferred to aCoplin jar containing deionized water. Deionized water was changed andincubated 60 seconds at room temperature.

The slides were dried in ethanol quickly by dipping them in Coplin jarscontaining 70%, 80% and 100% ethanol. The slides were removed and airdried. For long term storage, the slides were soaked in xylene, and adrop of Permount and a coverslip were added.

EXAMPLE 2 Detection of Single Gene Copies

The slides produced in Example 1 were mounted with Permount (FisherScientific, N.J.) and coverslipped.

Using brightfield microscopy, microphotographs were made on KodakEktachrome color slide film (EL 400), using a Zeiss Axiophot 20epi-fluorescence microscope (Zeiss, West Germany) equipped with a 100×Plan-Neofluar oil immersion objective or a 100× Plan-APO oil immersionobjection (Zeiss, West Germany), a Zeiss MC-100 camera (Zeiss, WestGermany), and a blue or neutral density filter.

As can be seen from FIGS. 1, 2 and 3 single blue signal was visible atthe location of each copy of the HER-2/neu gene. The expected resultsfor visualization of the HER-2/neu gene in normal metaphase chromosomesfrom the MDA-MB-468 breast tumor cell line is two; one on eachchromatin, and the number of the blue signals in normal interphasenuclei is also two, or possibly two pairs if the cell is in G2 phase. Inthe cultured breast tumor cell lines MDA-MB-361 and SK-BR-3, multipleblue signals are visible in both metaphase chromosomes and interphasenuclei, indicating presence of multiple copies of the HER-2/neu gene.The cell nuclei and chromosomes were stained light pink for colorcontrast.

EXAMPLE 3 Detection of High Risk Human Papilloma Virus in GynecologicTissue Specimen

Six separate commercially available plasmids, i.e., pGem2, pUC13, pGem1,pLINK322, pGem1 and pUCI3 containing entire genomes of HPV types 16 (DNAsequence available from GenBank, Accession No. K02718), 18 (DNA sequenceavailable from GenBank, Accession No. X05015), 31 (DNA sequenceavailable from GenBank, Accession No. J04353), 33 (DNA sequenceavailable from GenBank, Accession No. M12732), 35 (DNA sequenceavailable from GenBank, Accession No. M74117) and 51 (DNA sequenceavailable from GenBank, Accession No. M62877) respectively, were labeledby nick translation with digoxigenin dCTP. Alternatively, one may clonethe HPV into a plasmid by standard molecular biology techniques withinthe skill of the art. The labeled plasmids were then mixed together toform a single reagent. Incorporation of the digoxigenin nucleotide intothe labeled DNA was verified by dot-blot procedure. DNA fragment sizewas determined by gel electrophoresis and was ideally between 100 and200 base pairs.

Uterine cervix cells were sampled and smeared to form a conventional Papsmear or suspended in PreservCyt (Cytyc Corporation), a bufferedfixative and preservative solution. The ThinPrep 2000 (CytycCorporation) was used in make two ThinPrep slides for each patient. SeeLinder et al., The ThinPrep Pap Test, A Review of Clinical Studies, ActaCytologica, Vol. 41, No. 1, pp. 30-38 (1997) herein incorporated byreference. One slide was stained for conventional cytology similar tothat of conventional PAP smears and the other slide was prepared asbelow.

The Pap smear and the ThinPrep slide were incubated for 20 minutes at37° C. in a solution of 10 micrograms per milliliter of Proteinase K in2×SSC. Following the incubation, the slides were washed for 2 minutes atroom temperature in 2×SSC, dehydrated in a series of 70%, 80%, and 95%room temperature ethanol solutions for 1 minute each and air dried.

A probe solution was made using the above probes consisting of 0.5microgram per milliliter of HPV types 18, 33, 35, and 51 and 0.2micrograms per milliliter of HPV types 16 and 31 in Hybrisol VII (Oncor,Gaithersburg, Md.). Ten microliters of this probe solution was pipettedonto the ThinPrep sample slide and the specimen was covered with a 22 mmround coverslip and sealed with rubber cement. The slide was placed on aprewarmed 75° C. hot plate for 5 minutes to denature the probe andtarget DNA and then transferred to a humidified chamber and placed in a37° C. incubator.

The slide was incubated at 37° C. in the humidified chamber for 2 to 16hours to hybridize.

After the 37° C. incubation for hybridization, the rubber cement andcoverslip was removed. The slide was washed for 5 minutes at 72° C. inprewarmed 2×SSC. The slide was then transferred to a Coplin jar at roomtemperature containing 1×PBD (phosphate buffered detergent) forapproximately 2 minutes to equilibrate.

Both high stringency post hybridization wash conditions (0.2×SSC, 2%BSA, 60° C., 10 minutes) and low stringency post hybridization washconditions (2×SSC, 2% BSA, 45° C., 10 minutes) were used on a number ofpatient samples for whom the HPV type was determined.

The sample is incubated for 30 minutes at 37° C. with 100 μl of alkalinephosphatase (AP) labeled anti-digoxigenin antibody (Boehringer MannheimGMBH) diluted 1:150 in 100 mM Tris pH 7.5/150 mM NaCl. The slide iswashed three times for ten seconds using APK wash solution to remove anyunbound or loosely bound antibody.

The slide is removed and allowed to drain briefly. Four hundredmicroliters of Ventana Enhancer is added to the slide and the reactionproceeded at room temperature for 30-60 minutes while shaking. Theslides are rinsed in distilled water and allowed to air dry.

The slide is dipped in a ¼× solution of Eosin in ethanol tocounterstain. The slide is rinsed three times in distilled water andallowed to air dry. To mount, the slide is dipped in xylene and a dropof Permount (Fisher) is added. The slide is then covered with a 22 mmround glass coverslip.

Using brightfield microscopy, microphotographs are made on KodakEktachrome color slide film (EL 400), using a Zeiss Axiophot 20epi-fluorescence microscope (Zeiss, West Germany) equipped with a 100×Plan-Neofluar oil immersion objective or a 100× Plan-APO oil immersionobjection (Zeiss, West Germany), a Zeiss MC-100 camera (Zeiss, WestGermany), and a blue and/or neutral density filter.

Cells infected with HPV integration demonstrate a blue precipitate inthe nuclei with minimal slide background. The cytoplasm iscounterstained pink for contrast. Cellular morphology confirmed that HPVwas present in abnormal cells; normal cells do not exhibit a positivesignal.

A sizable number of normal, ASCUS and SIL PAP smears are tested for HPVtype status. Those detected by the probe reagent of the presentinvention are indicated as positive cases. The probes above are alsotested against three known cell lines having different copy numbers ofHPV to confirm the ability to detect HPV with respect to the copy numberof viruses in each cell line.

EXAMPLE 4 Automated ISH Staining and Single Gene Copy Detection

The paraffin-embedded method of Example 1 was repeated on a DISCOVERY™automated slide stainer described in PCT/US99/04181 with all steps beingperformed automatically. Using the same reagents and conditions ofExample 1, individual spots representing single gene copies wereobserved using the detection methods of Example 2.

Therefore, the above description should not be construed as limiting,but merely as exemplifications of preferred embodiments. Those skilledin the art will envision other modifications within the scope and spiritof the following claims.

All references mentioned above are incorporated in their entirety byreference.

1. A composition comprising at least one cell containing target nucleicacid sequences associated with a product of an enzyme and a chromogencomposition in the at least one cell, wherein the product isindividually associated with each of at least one of the target nucleicacid sequences and each of the target nucleic acid sequences associatedwith the product is separate from each other such that the product isdistinguishable from the product associated with other copies of thetarget nucleic acid sequences in the same cell such that one candetermine the number of original copies of the target nucleic acidsequences in the at least one cell.
 2. The composition according toclaim 1, wherein the product is located at the target nucleic acidsequence of a chromosomal DNA exclusively.
 3. The composition accordingto claim 1, wherein the enzyme is a phosphatase or a peroxidase.
 4. Thecomposition according to claim 1, wherein the chromogen is NBT/BCIP,tetramethylbenzidine or diamino benzidine.
 5. The composition accordingto claim 1 wherein the enzyme is alkaline phosphatase and the chromogenis NBT/BCIP.
 6. The composition according to claim 1, wherein the targetnucleic acid sequence is Her-2/neu DNA.
 7. The composition according toclaim 1, wherein the target nucleic acid sequence is a nucleic acidsequence selected from the group consisting of a nucleic acid sequenceof human immunodeficiency virus (HIV), human papilloma virus (HPV),Epstein-Barr virus (EBV), cytomegalovirus (CMV), T. palidium, and M.tuberculosis.
 8. The composition according to claim 1, wherein theenzyme is associated with a nucleic acid probe specific for the targetnucleic acid sequence and bound to the target nucleic acid sequence inthe at least one cell.
 9. The composition according to claim 8, whereinthe nucleic acid probe is bound to the target nucleic acid sequence viahybridization.
 10. The composition according to claim 8, wherein thenucleic acid probe is labeled with a detectable moiety.
 11. Thecomposition according to claim 10, wherein the detectable moiety isselected from the group consisting of digoxygenin, biotin andfluorescein.
 12. The composition according to claim 10, wherein theenzyme is linked to an antibody specific to the detectable moiety on thelabeled nucleic acid probe.
 13. The composition according to claim 12,wherein the enzyme is alkaline phosphatase; the detectable moiety isdigoxygenin; and the antibody is an antibody specific to digoxygenin andlinked to alkaline phosphatase.
 14. The composition according to claim1, wherein the product is distinguishable from the product associatedwith other copies of the target nucleic acid sequences in the same cellunder brightfield microscope conditions.
 15. A kit for detecting asingle gene copy target in an intact cell, comprising: a) adetectably-labeled nucleic acid probe specific for a Her-2/neu gene; b)an anti-digoxigenin antibody having an enzyme covalently attachedthereto; and c) a chromogen for deposition in the immediate vicinity ofthe enzyme-antibody complex.
 16. The composition according to claim 15,wherein the enzyme is a phosphatase or a peroxidase.
 17. The compositionaccording to claim 15, wherein the chromogen is NBT/BCIP,tetramethylbenzidine or diamino benzidine.
 18. The kit according toclaim 15, wherein the enzyme is alkaline phosphatase and the chromogenis NBT/BCIP.
 19. The kit according to claim 15, further comprising:instruction for detecting a single copy of the Her-2/neu gene in a cell.20. The kit according to claim 15, further comprising: a specimencontaining breast cells.